CN117064766B - Composite ROS (reactive oxygen species) responsive hydrogel as well as preparation method and application thereof - Google Patents
Composite ROS (reactive oxygen species) responsive hydrogel as well as preparation method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K6/00—Preparations for dentistry
- A61K6/15—Compositions characterised by their physical properties
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K6/00—Preparations for dentistry
- A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K6/00—Preparations for dentistry
- A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
- A61K6/884—Preparations for artificial teeth, for filling teeth or for capping teeth comprising natural or synthetic resins
- A61K6/887—Compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K6/00—Preparations for dentistry
- A61K6/80—Preparations for artificial teeth, for filling teeth or for capping teeth
- A61K6/884—Preparations for artificial teeth, for filling teeth or for capping teeth comprising natural or synthetic resins
- A61K6/898—Polysaccharides
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- Health & Medical Sciences (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Plastic & Reconstructive Surgery (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Preparation (AREA)
Abstract
The application relates to a composite ROS (reactive oxygen species) responsive hydrogel and a preparation method and application thereof, and belongs to the technical field of hydrogels. The preparation method of the composite ROS responsive hydrogel comprises the following steps: adding sodium alginate and small extracellular vesicles into the D-mannitol solution to obtain a sodium alginate solution containing the small extracellular vesicles; mixing the sodium alginate solution containing small extracellular vesicles, rhB-AC monomer solution and Ca 2+ The slow release agent, the cross-linking agent, the initiator and the catalyst are mixed for cross-linking reaction, and the composite ROS responsive hydrogel is obtained. The compound ROS response hydrogel is obtained by mixing sodium alginate solution containing small extracellular vesicles with RhB-AC monomer and cross-linking agent for cross-linking reaction, and has the advantages of good operability and biocompatibility and capability of slowly releasing the small extracellular vesicles.
Description
Technical Field
The application relates to the technical field of hydrogels, in particular to a composite ROS (reactive oxygen species) responsive hydrogel and a preparation method and application thereof.
Background
When infection and inflammatory reaction occur in the dental pulp of the permanent tooth due to the stimulation of bacteria, machinery, chemistry and the like, the most common treatment regimen is root canal treatment, but the treatment regimen fails to retain dental pulp with nutrition, perception, immunity and restoration functions, and the technical sensitivity and treatment cost are high. In recent years, along with development of the concept of minimally invasive treatment of dental pulp and update of treatment means, live pulp preservation treatment is gradually becoming another viable treatment scheme for pulpitis. The preservation treatment of the constant dental pulp covers bioactive pulp covering materials on the surface of the near pulp dentin or the exposed dental pulp wound surface by removing the infected hard tissues and the affected dental pulp so as to eliminate lesions and promote the healing of the damaged dental pulp. However, the current pulp capping preparation is mainly suitable for healthy pulp wounds and has poor curative effect on inflammatory pulp. The research and development of the novel pulp capping material for the preservation of the living pulp of the inflammatory dental pulp is expected to improve the clinical curative effect of the preservation treatment of the living pulp, the preservation of the healthy dental pulp is maximized, and the novel pulp capping material has important significance for improving the life quality of patients and reducing the treatment cost of dental pulp diseases.
Disclosure of Invention
The application aims to overcome the defects of the prior art and provide a compound ROS response type hydrogel capable of slowly releasing small extracellular vesicles, and a preparation method and application thereof.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides a method of preparing a composite ROS-responsive hydrogel comprising the steps of:
(1) Adding sodium alginate and small extracellular vesicles into the D-mannitol solution to obtain a sodium alginate solution containing the small extracellular vesicles; the small extracellular vesicles are derived from odontocyst stem cells;
(2) Mixing the sodium alginate solution containing small extracellular vesicles in the step (1), rhB-AC monomer and Ca 2+ The slow release agent, the cross-linking agent, the initiator and the catalyst are mixed for cross-linking reaction, so that the composite ROS responsive hydrogel is obtained; the mass percentage of the RhB-AC monomer in the RhB-AC monomer solution is 8.5-10%; the structural formula of the RhB-AC monomer is shown as the following formula:
according to the preparation method, the sodium alginate solution containing the small extracellular vesicles is mixed with the RhB-AC monomer and the auxiliary agent for crosslinking reaction, so that the compound ROS response hydrogel which is good in operability and biocompatibility and capable of slowly releasing the small extracellular vesicles is obtained.
The small extracellular vesicles (sEV) are derived from Dental Follicle Stem Cells (DFSCs), the dental follicle stem cells are easily obtained dental mesenchymal stem cells, have the effect of promoting inflammatory dental pulp repair, sEV is an important effector molecule of the stem cells for playing a paracrine role, so sEV in the DFSCs is extracted through multi-step separation, and in-vitro experiments prove that sEV derived from the DFSCs has the effect of relieving H 2 O 2 Induced Dental Pulp Stem Cells (DPSCs) oxidative stress, reduced apoptosis, enhanced cell proliferation, and the ability to repair the odontoblast differentiation of DPSCs; in experimental pulpitis models from DFSC-sEV as a pulp capping material to rats, experimental results show that DFSC-sEV can relieve dental pulp oxidative stress injury and promote the formation of a wound dentin bridge, and the results show that DFSC-sEV can be used as a novel pulp capping material for promoting the repair of inflammatory dental pulp. However, during the course of the experiment, the applicant found that sEV was easily cleared by macrophages when applied in vivo, and had the disadvantage of unstable therapeutic effects. On this basis, the applicant overcomes the instability defect of sEV in vivo application by loading sEV into hydrogels with ROS response characteristics, causing the hydrogels to release sEV "on demand" in environments with higher ROS levels.
The RhB-AC monomer is a compound containing a polymerized double bond and can respond to HClO/ClO in the environment - While HClO/ClO - Belonging to one of the ROS, when the ROS level in the environment reaches a certain threshold (e.g. HClO/ClO) - At a concentration of 50 μm), the polymeric double bonds in the RhB-AC monomer are dissociated, achieving an effect of responding to ROS.
As a preferred embodiment of the preparation method described herein, in the step (1), the mass ratio of the sodium alginate, the small extracellular vesicles, and the D-mannitol in the D-mannitol solution is: small extracellular vesicles: d-mannitol = 2:0.0003:1;
in step (2)The Ca is 2+ The volume ratio of the sum of the volumes of the sustained release agent, the cross-linking agent, the initiator and the catalyst to the sodium alginate solution containing small extracellular vesicles, rhB-AC monomer solution is Ca 2+ Sustained release agent + crosslinking agent + initiator + catalyst: sodium alginate solution containing small extracellular vesicles: rhB-AC monomer solution=317.5:2700:25.
Under the condition of preferable proportion, the compound ROS response hydrogel has relatively long gel forming time, can form gel within 4min, endows the hydrogel with good injectability, and has uniform shape, white semitransparent shape and porous and network internal structure, thereby being beneficial to load sEV. Ca is selected for the application 2+ The slow release agent can promote sodium alginate to coagulate into a network, and the crosslinking agent, the initiator and the catalyst are added to help to generate free radicals to initiate the polymerization of the RhB-AC monomer to form a network structure, and then the network structure is polymerized with the sodium alginate network to obtain the compound ROS responsive hydrogel.
As a preferred embodiment of the preparation method described herein, in the step (2), the mass fraction ratio of the crosslinking agent, the initiator and the catalyst is that of the crosslinking agent: and (3) an initiator: catalyst = 0.164:0.164:0.008; the cross-linking agent is N, N' -methylene bisacrylamide; the initiator is ammonium persulfate; the catalyst is tetramethyl ethylenediamine. The ammonium persulfate generates free radicals under the action of tetramethyl ethylenediamine to initiate the polymerization of the RhB-AC monomer, and the RhB-AC polymer is crosslinked with sodium alginate under the action of N, N' -methylene bisacrylamide to form the composite ROS responsive hydrogel.
As a preferred embodiment of the production method described herein, the Ca 2+ The slow release agent comprises soluble calcium salt and disodium hydrogen phosphate, wherein Ca in the soluble calcium salt 2+ Molar concentration ratio with disodium hydrogen phosphate of Ca 2+ : disodium hydrogen phosphate=0.08:0.0537. Under the condition of preferable proportioning, the combination of the soluble calcium salt and the disodium hydrogen phosphate is Ca 2+ The slow release agent can lead sodium alginate solution and Ca 2+ The speed of ion exchange reaction is slowed down, the forming speed of the compound ROS response type hydrogel is controlled to be about 4min, and the compound ROS response type hydrogel is usedCan be applied to the wound surface in the process by injection, and can be glued without waiting for a long time.
As a preferred embodiment of the preparation method described herein, in step (1), the small extracellular vesicles are extracted according to the following method:
s1, inoculating odontoblast stem cells into a serum-free culture medium for culture, removing cell fragments, and centrifuging to obtain a supernatant a;
s2, performing ultracentrifugation on the supernatant a obtained in the step S1 to obtain a precipitate a; the rotating speed of the ultracentrifugation is 100000-120000g;
s3, re-suspending the sediment a in the step S2 by adopting PBS, washing and centrifuging to obtain sediment b, wherein the sediment b is small extracellular vesicles.
sEV is extracted from the dental follicle stem cells by multi-step centrifugation, and the precipitate obtained by the extraction method is determined to be the small extracellular vesicles through a series of markers for identifying the small extracellular vesicles. In addition, the applicant also determines through biological experiments that sEV extracted from the odontoblast stem cells can improve H 2 O 2 Mediated oxidative and antioxidant imbalance of DPSCs, reduction of apoptosis and proliferation inhibition, restoration of odontoblast differentiation ability of DPSCs in oxidative stress environment, and sEV is suggested to have the effect of relieving H 2 O 2 Induced dental pulp stem cell oxidative stress injury.
As a preferred embodiment of the preparation process described herein, in step (2), the crosslinking reaction is stirred at 20 to 30℃for 30 to 40 minutes. The preparation method of the composite ROS responsive hydrogel is quick and convenient, the crosslinking reaction can be realized only by stirring under the room temperature condition, and the harsh reaction condition and longer waiting time are not needed.
In a second aspect, the present application provides a composite ROS responsive hydrogel prepared according to the preparation method described above.
In a third aspect, the application provides an application of the composite ROS responsive hydrogel in preparing a material for treating pulpitis or promoting wound repair.
The application of the composite ROS responsive hydrogel as a repairing material in the treatment of pulpitis can intelligently and slowly release sEV according to the ROS level in inflammatory pulps, so that the composite ROS responsive hydrogel plays roles in protecting oxidative stress damaged DPSCs, promoting the differentiation of DPSCs odontoblasts, forming dentin-like barriers on the dental pulp wound surface, and realizing the preservation and repair of inflammatory pulps.
As a preferred embodiment of the application described herein, the material is one of a wound dressing, a filler, a medullary material.
In a fourth aspect, the present application provides a bio-medullary material comprising the composite ROS-responsive hydrogel described above.
Compared with the prior art, the beneficial effects of this application are:
(1) According to the preparation method, the small extracellular vesicles (sEV) are loaded into the hydrogel capable of responding to the ROS to prepare the compound ROS-responsive hydrogel, after the ROS level of the environment where the compound ROS-responsive hydrogel is located is raised, the compound ROS-responsive hydrogel can rapidly and sensitively respond to the ROS and is degraded, sEV is released from the hydrogel into the environment, the effect of slowly releasing sEV is achieved, and the effect of promoting repair and preservation of inflammatory dental pulp is achieved.
(2) The compound ROS response hydrogel is obtained through the cross-linking reaction of the sodium alginate solution containing the small extracellular vesicles, the RhB-AC monomer and the cross-linking agent, the sodium alginate and the small extracellular vesicles are cross-linked into a layer of network under the action of the cross-linking agent, the cross-linking agent and the RhB-AC are polymerized into a second layer of network, the small extracellular vesicles can be uniformly distributed, sEV is prevented from being clearly formed by macrophages in a body, and meanwhile, according to the ROS level of the environment, the intelligent slow release sEV is realized, and the protection and restoration effect of sEV on inflammatory dental pulp is enhanced.
Drawings
FIG. 1 shows characterization and biological identification results of small extracellular vesicles (sEV) of the application, wherein A is an extracted sEV transmission electron microscope image, B is an extracted sEV diameter distribution map, and C is a sEV specific marker detection result;
FIG. 2 shows the passage of the present application through different concentrations of H 2 O 2 Solution treatment of Dental Pulp Stem Cells (DPSCs) to create a result graph of a DPSCs in vitro oxidative stress model;
FIG. 3 is a graph showing the effect of sEV extracted in the application on a DPSCs in-vitro oxidative stress model, wherein A is the result of DPSCs intracellular Reactive Oxygen Species (ROS) horizontal flow fluorescence detection, B is the result of DPSCs intracellular ROS level semi-quantification, C is the glutaraldehyde (MDA) expression level of DPSCs, D is the superoxide dismutase (SOD) activity level of DPSCs, E is the result of DNA oxidative damage marking 8-OHDG semi-quantification in DPSCs, and F is the DNA oxidative damage marking 8-OHDG distribution map in DPSCs;
FIG. 4 is a graph of the effect of sEV on DPSCs in vitro oxidative stress model, wherein A is a FITC Annexin V/7AAD staining relation analysis graph of DPSCs, B is apoptosis rate of DPSCs, C is a fluorescence microscope graph for evaluating proliferation capacity of DPSCs by BrdU staining method, and D is BrdU positive cell proportion of DPSCs;
FIG. 5 is a graph showing the effect of sEV extracted in the present application on an in vitro oxidative stress model of DPSCs, wherein A is alizarin red staining for evaluating mineralization nodule formation level of DPSCs, B is related gene expression level of DPSCs odontoblasts to differentiation markers, C is oil red O staining for evaluating lipid drop formation level of DPSCs, and D is related gene expression level of DPSCs lipid to differentiation markers;
FIG. 6 is a representation of data obtained from the composite ROS-responsive hydrogel of the present application, wherein A is a structural microscopic image of the composite ROS-responsive hydrogels of example 1, example 2, comparative example 1, and comparative example 2, B is a synthetic schematic representation of the composite ROS-responsive hydrogel, C is a composite ROS-responsive hydrogel swelling property assessment, and D is the gel formation time of the composite ROS-responsive hydrogel;
FIG. 7 is a graph showing the results of load and in vitro sustained release sEV performance measurements of a composite ROS-responsive hydrogel of the present application, wherein A is the ratio of the composite ROS-responsive hydrogel to HClO/ClO - B is the evaluation result of the degradation characteristics of the composite ROS responsive hydrogel under different solution environments, C is the distribution condition of sEV in the composite ROS responsive hydrogel, and D is the distribution condition of the composite ROS responsive hydrogel in HClO/ClO - As a result of the slow release of sEV in the solution, E is the effect of the degradation product of the composite ROS-responsive hydrogel on the proliferation activity of DPSCs, and F is the effect of the degradation product of the composite ROS-responsive hydrogel on the activity of DPSCs;
FIG. 8 is a graph showing the effect of the composite ROS-responsive hydrogel of the present application on treatment of inflammatory dental pulp for 3 days, wherein A is the level of dental pulp oxidative stress damage, B is a semi-quantitative analysis of 8-OhdG expression levels, and C is CD90 of DPSCs + Analyzing the degree of oxidative stress damage;
FIG. 9 is a graph showing the effect of the composite ROS-responsive hydrogel of the present application on treatment of inflammatory dental pulp for 7 days, wherein A is the result of DPSS immunofluorescence evaluation of dental pulp mineralization restoration level, and B is the result of semi-quantitative analysis of dental pulp DPSS level;
FIG. 10 is a graph showing the strength of hard tissue barrier formation of dental pulp wound surface 28 days after treatment of inflammatory dental pulp with the composite ROS-responsive hydrogel of the present application;
FIG. 11 is a schematic illustration of the mechanism of action of the composite ROS responsive hydrogel of the present application;
in the above figures, MFI is represented as mean fluorescence intensity, control is a blank Control group, SA-RhB is the composite ROS-responsive hydrogel of comparative example 1, SA-rhb@sev is the composite ROS-responsive hydrogel of example 1, LPS is the inflammation inducer lipopolysaccharide, P < 0.05, P < 0.01, and P < 0.001.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to specific examples.
Materials, reagents and the like used in the examples, comparative examples and experimental examples were commercially available unless otherwise specified.
The following examples, comparative examples and experimental examples were prepared by extracting dental follicle stem cells, dental pulp stem cells and small extracellular vesicles by the following methods:
isolation culture of bursa stem cells (DFSCs): taking a rat with the birth of 4-6d, performing blunt separation on mandible after dislocation of cervical vertebra, separating molar tooth embryo under a microscope, peeling tooth embryo outer tooth bag tissue, washing tooth bag tissue for 2 times by using an alpha-MEM culture medium, uniformly spreading the tooth bag tissue in a culture dish wetted by a primary culture medium after shearing, culturing overnight, adding the primary culture medium, performing subculture every 3d of replacement liquid when cells grow to 80-90% confluence, obtaining tooth bag stem cells, identifying vitality of the tooth bag stem cells, including marker detection, dentin differentiation capability detection and adipogenic differentiation capability, wherein the markers comprise CD29, CD45, CD90, CD34 and CD44, and 3 rd-5 th generation tooth bag stem cells which can detect the markers and have the dentin differentiation capability and the adipogenic differentiation capability can be used for subsequent experiments.
The isolated culture method of Dental Pulp Stem Cells (DPSCs) is similar to that of dental follicle stem cells, except that the cells are derived from rats of 4-6 weeks, the isolated tissue is dental pulp, and the rest of the operations are unchanged.
Preparation of small extracellular vesicles (sEV):
s1, inoculating odontoblast stem cells into a serum-free culture medium for culturing for 48 hours, centrifuging the culture solution at 3000g for 15min to remove cell fragments, and obtaining a supernatant a;
s2, centrifuging the supernatant a obtained in the step S1 at 120000g for 90min to obtain a precipitate a;
s3, re-suspending the sediment a in the step S3 by using PBS, washing and centrifuging to obtain sediment b, and detecting that the sediment b is a circular vesicle with the diameter of 30-200nm and has the activities of TSG101, CD63 and CD9, thus being identified as the small extracellular vesicle.
Preparation of RhB-AC monomer: reducing rhodamine B with hydrazine and methanol to obtain RhB-NH 2 And then the RhB-NH is added 2 And carrying out substitution reaction with triethylamine, dichloromethane and acryloyl chloride at 0 ℃ to obtain the RhB-AC monomer.
Example 1
According to one embodiment of the composite ROS responsive hydrogel and the preparation method thereof, the composite ROS responsive hydrogel is prepared by the following steps:
(1) 2g of sodium alginate and 0.0003g of small extracellular vesicles are added into 100mL of 1wt% D-mannitol solution, and uniformly mixed to obtain sodium alginate solution containing the small extracellular vesicles;
(2) 2.7mL of the sodium alginate solution containing small extracellular vesicles from step (1), 0.025mL of 10wt% RhB-AC monomer solution, and 0.275mL of Ca 2+ Sustained release agent, 0.02mL 0.164wt%N,N' -methylenebisacrylamide, 0.02mL of 0.164wt% ammonium persulfate and 0.0025mL of 0.008wt% tetramethylethylene glycolMixing amine, stirring for 30-40min at room temperature to perform crosslinking reaction to obtain composite ROS responsive hydrogel, and designating the obtained hydrogel as SA-RhB@sEV; the Ca is 2+ The slow release agent comprises 0.272M calcium sulfate and 0.054M disodium hydrogen phosphate.
Example 2
In one embodiment of the compound ROS responsive hydrogel and the preparation method thereof, the preparation method of the compound ROS responsive hydrogel is similar to that of the embodiment 1, except that the small extracellular vesicles in the step (1) are 0.0006g, the 8.5wt% RhB-AC monomer solution in the step (2) is 0.03mL, and the rest steps and parameters are unchanged, so that the obtained hydrogel is named SA-RhB@sEV1.
Example 3
In one embodiment of the compound ROS responsive hydrogel and the preparation method thereof, the preparation method of the compound ROS responsive hydrogel is similar to that of the embodiment 1, except that the small extracellular vesicles in the step (1) are 0.00045g, the 9.25wt% RhB-AC monomer solution in the step (2) is 0.0275mL, and the rest steps and parameters are unchanged, and the obtained hydrogel is named SA-RhB@sEV2.
Comparative example 1
The preparation method of the compound ROS-responsive hydrogel is similar to that of example 1, except that RhB-AC monomer is not added in step (2), and the other steps and parameters are unchanged, and the obtained hydrogel is named SA.
Comparative example 2
The preparation method of the compound ROS-responsive hydrogel is similar to that of example 1, except that the amount of 8.5wt% of RhB-AC monomer solution in step (2) is 0.06mL, and the rest steps and parameters are unchanged, so that the obtained hydrogel is named SA-RhB@sEV3.
Comparative example 3
The preparation method of the compound ROS-responsive hydrogel is similar to that of example 1, except that small extracellular vesicles are not added in step (1), and the other steps and parameters are unchanged, so that the obtained hydrogel is named SA-RhB.
Experimental example 1
Dental Pulp Stem Cells (DPSCs) are mesenchymal stem cells derived from dental pulp tissues and are key factors for repairing damaged dental pulp, and researches show that dental pulp inflammatory tissues have oxidative stress characteristics, are shown as abnormal rise of Reactive Oxygen Species (ROS) levels, cause apoptosis of the DPSCs due to oxidation and antioxidation imbalance, inhibit the multi-directional differentiation capacity of the DPSCs, seriously weaken the tissue repairing or regenerating function mediated by the DPSCs, and are helpful for repairing the damage of inflammatory dental pulp if the adaptability of the DPSCs to oxidative stress environments can be enhanced. Therefore, the experiment judges sEV the influence of DPSCs in the oxidative stress state by establishing an in vitro oxidative stress model of the DPSCs and adopting sEV treatment to measure the related indexes of the DPSCs, and searches sEV whether the effect of repairing inflammatory dental pulp can be realized by influencing the DPSCs.
1. And (5) characterizing and biologically identifying the extracted odontoblast stem cell source sEV.
Detecting sEV total protein concentration of the extracted sEV according to the BCA protein concentration determination kit instructions; detecting the morphology and size distribution of sEV by using a transmission electron microscope and a Zeta View nanoparticle tracking analyzer; the Western blot was used to determine whether specific markers TSG101, CD63 and CD9 were expressed in sEV, as shown in FIG. 1.
As shown in FIG. 1, the total particle concentration of sEV obtained by extraction is (1.33.+ -. 0.09). Times.10 8 particles/ml, is in a round and double-layer membrane-like structure, has an average particle size of 163.12 +/-4.07 nm and a peak value of 125nm, and is in the particle size range of small extracellular vesicles; and TSG101, CD63 and CD9 can be specifically expressed, which shows that the method can successfully extract sEV with better activity.
2. By H 2 O 2 And establishing a DPSCs in-vitro oxidation stress model.
3 rd generation DPSCs are processed into 3×10 3 Inoculating the cells/well into 96-well cell culture plate, culturing overnight until it adheres to the wall, and adding H with final concentration of 0, 50, 100, 200, 300, 500, 700 μm respectively 2 O 2 After 6 hours of culture, the solution was changed to subculture medium and cultured for 24 hours, DPSCs proliferation activity was evaluated by using CCK8 kit, and the results are shown in FIG. 2.
As shown in FIG. 2, 200. Mu.M H 2 O 2 The solution was able to reduce the viability of the DPCS to 60% of normal cells, thus 200. Mu.M H was used 2 O 2 Solution treatment DPCSs were used as in vitro oxidative stress models for DPSCs.
3. The change in the in vitro oxidative stress model of DPSCs treated with sEV was evaluated.
To facilitate the observation, PKH-67 green fluorescent dye was used to label sEV, and the remainder of the experiments were labeled sEV unless otherwise specified. The experiment is provided with 3 treatment groups, namely a blank control group and H 2 O 2 Group sum H 2 O 2 Group + sEV. Blank control group does not do H 2 O 2 Solution treatment; h 2 O 2 Group sum H 2 O 2 200. Mu.M H was added to the + sEV groups 2 O 2 Solution stimulation for 6h, H 2 O 2 Group + sEV in addition of H 2 O 2 The solution was pretreated with sEV at 40. Mu.g/mL for 24h.
Will warp H 2 O 2 The three treatment groups after solution treatment respectively adopt a flow cytometry to detect ROS level, 8-OHdG staining to evaluate the DNA oxidative damage degree of the cells, detect ROS, MDA, SOD of the cells and apoptosis level according to the steps of a kit, evaluate the proliferation capacity of the cells by a BrdU method, evaluate the dentinogenesis/adiposity differentiation capacity of the cells by qPCR and alizarin red staining/oil red O staining, and the results are shown in figures 3-5.
As shown in FIGS. 3-A and 3-B, H 2 O 2 Cellular ROS levels in the + sEV group were lower than H 2 O 2 The ROS level of the group is close to that of a blank control group, which shows that sEV can effectively reduce the ROS level of a DPSCs in-vitro oxidative stress model; as shown in FIGS. 3-C, 3-D, 3-E, 3-F, H 2 O 2 The cellular MDA, SOD level and DNA oxidative damage degree of + sEV group are all lower than H 2 O 2 The group, close to the blank group, showed that sEV was effective in reducing MDA and SOD levels, and DNA oxidative damage levels of the DPSCs in vitro oxidative stress model.
As shown in FIGS. 4-A and 4-B, H 2 O 2 Apoptosis level in + sEV group was lower than H 2 O 2 Group, close to the blank group, showed sEV to reduce apoptosis levels of DPSCs in vitro oxidative stress model; as shown in FIGS. 4-C and 4-D, H 2 O 2 The cell proliferation capacity of + sEV group was higher than that of H 2 O 2 The group, close to the blank group, showed that sEV was able to increase the proliferation capacity of the DPSCs in vitro oxidative stress model.
As shown in FIGS. 5-A and 5-B, H 2 O 2 Mineralization nodule increase, cellular mineralization markers ALP, BSP and DCN levels were higher than H in + sEV group 2 O 2 Group, lower than the blank group, showing sEV is able to promote the odontoblast differentiation ability of DPSCs in vitro oxidative stress model; as shown in FIGS. 5-C and 5-D, H 2 O 2 The level of cell adipogenic related genes Adipsin, CEBP alpha and CEBP beta in + sEV group is lower than H 2 O 2 Group, close to the blank group, showed sEV to restore the adipogenic differentiation ability of DPSCs in vitro oxidative stress model to normal cell levels.
From the above data, sEV extracted from DFSC can improve H 2 O 2 Mediated oxidative and antioxidant imbalance of DPSCs, reduced apoptosis and proliferation inhibition, and restoration of odontoblast differentiation capability of DPSCs under oxidative stress environment, i.e. sEV with H relieving effect 2 O 2 Effect of induced oxidative stress loss of DPSCs.
Experimental example 2
The composite ROS-responsive hydrogels were characterized and the loading and in vitro release sEV performance of the hydrogels were evaluated.
1. The composite ROS-responsive hydrogels were characterized.
(1) Observing the internal morphology of the composite ROS-responsive hydrogels of examples 1-3 and comparative examples 1-2 by a scanning electron microscope;
(2) The hydrogel of example 1 was immersed in PBS and weighed at regular time to calculate the swelling ratio of the hydrogel;
the above results are shown in FIG. 6.
2. Hydrogel loading and in vitro release sEV performance were evaluated.
(1) The hydrogel of example 1 was immersed in 0, 50, 100, 200, 500, 1000. Mu.M HClO/ClO - In solution, hydrogel pair HClO/ClO was evaluated by fluorescence spectroscopy - Is a response capability of (1);
(2) The hydrogels of example 1 were immersed in 100, 500, 1000. Mu.M HClO/ClO in PBS, respectively - Solution, 500. Mu.M HClO/ClO - In +10mM EDTA solution and 20mM EDTA solution, observing the degradation condition of the hydrogel, and obtaining the degraded hydrogel;
(3) The hydrogels of example 1 and comparative example 3 were photographed using a laser confocal microscope and three-dimensional images of the hydrogels were reconstructed and the distribution of sEV in the hydrogels was observed;
(4) The hydrogel of example 1 was immersed in 0, 100, 500, 1000. Mu.M HClO/ClO - In the solution, incubating at 100rpm in a shaking table at 37 ℃, and replacing the solution at 0, 0.5,1,2,3,4 and 5d, measuring the protein concentration of the replaced solution by a BCA method, calculating sEV cumulative release percentage P until the protein content in the solution is lower than the detection threshold of the BCA, wherein the calculation formula of sEV cumulative release percentage is as follows:
P＝(∑Ct)×2/300×100％
wherein Ct is the protein concentration at different times, t is time (days), t= 0.5,1,2,3,4,5;
(5) Co-culturing the degraded hydrogel with DPSCs, and evaluating the influence of hydrogel degradation products on the DPSCs through CCK8 and Live/read staining;
the above results are shown in FIG. 7.
As shown in fig. 6-a and 6-B, the structure of the compound ROS-responsive hydrogel of examples 1-3 of the present application is that RhB-AC forms an interpenetrating network structure with sodium alginate, the inside is a porous, network-like structure, and the inner wall surface is coarser than comparative example 1 and the degree of crosslinking increases, suggesting that the hydrogel forms a second network layer; the lower degree of cross-linking of the hydrogels of comparative example 2 compared to examples 1-3 indicates that the present application can provide a composite ROS-responsive hydrogel with higher degree of cross-linking, a rougher inner wall surface, and more sEV. As shown in FIGS. 6-C and 6-D, the composite ROS-responsive hydrogel of the present application can gel within 4min, is a uniform, white translucent gel, and has a swelling ratio of about 20% in PBS.
As shown in FIG. 7-A, the composite ROS-responsive hydrogel of the present application is capable of rapidly and sensitively responding to HClO/ClO - The solution emits fluorescence, and the fluorescence intensity is along with HClO/ClO - Increasing the concentration and increasing the contact time; as shown in FIG. 7-B, the composite ROS-responsive hydrogel of the present application is degradable, can degrade in different solutions, and the degradation rate follows HClO/ClO - The concentration increases and accelerates; as shown in FIGS. 7-C, 7-D, sEV in the composite ROS-responsive hydrogels of the present application are uniformly and densely distributed in the hydrogel and respond to HClO/ClO when the hydrogel is in contact with the hydrogel - Can continuously release sEV when in solution, and the release rate and HClO/ClO - The concentration of the compound ROS responsive hydrogel is in positive correlation, and the compound ROS responsive hydrogel is in 1000 mu M HClO/ClO - Action 5d in solution may release about 80% of sEV; as shown in FIGS. 7-E and 7-F, the composite ROS-responsive hydrogels of the present application were not significantly cytotoxic to DPSCs.
From the above data, the composite ROS-responsive hydrogel of the present application has good handleability, biocompatibility, and sEV sustained release properties.
Experimental example 3
To verify the therapeutic effect of the composite ROS-responsive hydrogel in pulpitis, experimental pulpitis of rats is established and directly covered, and the relevant indexes are measured to evaluate the curative effect of the composite ROS-responsive hydrogel on pulpitis.
1. Establishing an pulpitis model: anesthetizing rats with 3%H 2 O 2 The solution disinfects the oral cavity, the 2.5% NaClO solution disinfects the first molar crown, the mesial fossa of the occlusal surface of the first molar of the upper jaw on both sides of the dental crown is pulped, the wound surface is washed by 2.5% NaClO until the exposed pulp point has no obvious blood seepage, then the wound surface is thoroughly washed by PBS, and 5mg/mL Lipopolysaccharide (LPS) is injected into the exposed pulp point to induce pulpitis.
2. The osteosynthesis was performed using ROS-responsive hydrogels: the medicines of different treatment groups are gently placed on the dental pulp wound surface, then the wound surface is sealed by iRoot BP Plus, the cavity is wiped dry, the cavity is bonded by coating self-etching adhesive, the defect of the tooth body is repaired by using fluid resin, the occlusal surface of the first molar of the mandible is adjusted, and the filling body is prevented from falling off. The treatment groups included a blank, LPS, LPS+SA-RhB, LPS+ sEV, LPS+SA-RhB@sEV.
3. The degree of dental pulp inflammation and repair of the rats after the operation was evaluated by HE staining, the degree of oxidative stress damage of DPSCs in dental pulp and dental pulp of the rats after the operation was evaluated by 8-OHdG/CD105 immunofluorescence staining, and the dental pulp mineralization repair level of the rats after the operation was evaluated by DSPP immunofluorescence staining, and the results are shown in FIGS. 8, 9 and 10.
As shown in fig. 8-a, 8-B, 8-C, the post-operative 3d lps+sa-RhB, lps+ sEV, and lps+sa-rhb@sev groups had significantly reduced levels of 8-OHdG expression and 8-ohdg+cd90+ DPSCs ratios in dental pulp tissue compared to the LPS group, with 8-OHdG levels in the lps+sa-rhb@sev group significantly lower than in the rest of the treatments.
As shown in fig. 9-a, 9-B, post-operative 7d lps+sev and lps+sa-rhb@sev groups had significantly up-regulated levels of DSPP expression of the dental pulp mineralization repair markers compared to the remaining treatment groups, and the levels of DSPP expression were higher in the lps+sa-rhb@sev group than in the lps+ sEV group.
As shown in fig. 10, the wound surfaces of the post-operation 28d lps+sev group and the lps+sa-rhb@sev group all showed continuous dentin bridge formation, and the dentin bridge formation condition of the lps+sa-rhb@sev group was significantly better than that of the lps+ sEV group.
From the data, the compound ROS response hydrogel can respond to ROS intelligent slow release sEV in vivo, and the protective and repairing effects of sEV on inflammatory dental pulp are further enhanced.
The present application is drawn as a schematic representation of the mechanism of action of the composite ROS-responsive hydrogel according to the above results (fig. 11). As shown in fig. 11, when the dental pulp is inflamed, the DPSCs can generate oxidative stress phenomenon, the ROS level of the cells is increased, and the compound ROS-responsive hydrogel of the present application can start to degrade in an environment with a higher ROS level, so that sEV inserted in the hydrogel is slowly released into the dental pulp, the DPSCs damaged by oxidative stress are protected, the differentiation of the DPSCs into dentin cells is promoted, and dentin-like barriers are formed on the dental pulp wound surface, thereby realizing the preservation and repair of the inflamed dental pulp.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present application and not for limiting the scope of protection of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.
Claims (8)
1. The preparation method of the composite ROS responsive hydrogel is characterized by comprising the following steps of:
(1) Adding sodium alginate and small extracellular vesicles into the D-mannitol solution to obtain a sodium alginate solution containing the small extracellular vesicles; the small extracellular vesicles are derived from odontocyst stem cells;
(2) Mixing the sodium alginate solution containing small extracellular vesicles, rhB-AC monomer solution and Ca obtained in the step (1) 2+ The slow release agent, the cross-linking agent, the initiator and the catalyst are mixed for cross-linking reaction, so that the composite ROS responsive hydrogel is obtained; the mass percentage of the RhB-AC monomer in the RhB-AC monomer solution is 8.5-10%; the structural formula of the RhB-AC monomer is shown as the following formula:
；
in the step (1), the mass ratio of the sodium alginate to the small extracellular vesicles to the D-mannitol in the D-mannitol solution is as follows: small extracellular vesicles: d-mannitol = 2:0.0003-0.0006:1;
in step (2), the Ca 2+ The volume ratio of the sum of the volumes of the sustained release agent, the cross-linking agent, the initiator and the catalyst to the sodium alginate solution containing small extracellular vesicles, rhB-AC monomer solution is Ca 2+ Sustained release agent + crosslinking agent + initiator + catalyst: sodium alginate solution containing small extracellular vesicles: rhB-AC monomer solution = 317.5:2700:25-30;
in the step (2), the mass ratio of the cross-linking agent to the initiator to the catalyst is as follows: and (3) an initiator: catalyst = 0.164:0.164:0.008; the cross-linking agent is N, N' -methylene bisacrylamide; the initiator is ammonium persulfate; the catalyst is tetramethyl ethylenediamine.
2. The method of claim 1, wherein the Ca 2+ The slow release agent comprises soluble calcium salt and disodium hydrogen phosphate, wherein Ca in the soluble calcium salt 2+ Molar concentration ratio with disodium hydrogen phosphate of Ca 2+ : disodium hydrogen phosphate=0.08:0.0174.
3. The method of claim 1, wherein in step (1), the small extracellular vesicles are extracted according to the following method:
s1, inoculating odontoblast stem cells into a serum-free culture medium for culture, and removing cell fragments to obtain a supernatant a;
s2, performing ultracentrifugation on the supernatant a obtained in the step S1 to obtain a precipitate a; the rotating speed of the ultracentrifugation is 100000-120000g;
s3, re-suspending the sediment a in the step S2 by adopting PBS, washing and centrifuging to obtain sediment b, wherein the sediment b is small extracellular vesicles.
4. The method according to claim 1, wherein in the step (2), the crosslinking reaction is stirred at 20 to 30℃for 30 to 40 minutes.
5. A composite ROS-responsive hydrogel prepared according to the method of any one of claims 1-4.
6. The use of the composite ROS-responsive hydrogel of claim 5 in the preparation of a material for treating pulpitis or promoting repair of dental pulp wounds.
7. The use of claim 6, wherein the material is one of a wound dressing, a filler, a medullary covering material.
8. A bio-medullary material comprising the composite ROS-responsive hydrogel of claim 6.
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| CN111748520A (en) * | 2020-06-30 | 2020-10-09 | 四川大学 | Tooth sac stem cell exosome, preparation method and application thereof, composition thereof and preparation method |
| CN115137694A (en) * | 2022-07-15 | 2022-10-04 | 上海市第一人民医院 | Hydrogel material for spinal cord injury and preparation method and application thereof |
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| CN115569111A (en) * | 2021-10-26 | 2023-01-06 | 江阴司特易生物技术有限公司 | Composition comprising mesenchymal stem cells and hydrogel and use thereof |
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| CN111748520A (en) * | 2020-06-30 | 2020-10-09 | 四川大学 | Tooth sac stem cell exosome, preparation method and application thereof, composition thereof and preparation method |
| CN115569111A (en) * | 2021-10-26 | 2023-01-06 | 江阴司特易生物技术有限公司 | Composition comprising mesenchymal stem cells and hydrogel and use thereof |
| CN115137694A (en) * | 2022-07-15 | 2022-10-04 | 上海市第一人民医院 | Hydrogel material for spinal cord injury and preparation method and application thereof |
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